Enhanced binding of an HU homologue under increased DNA supercoiling preserves chromosome organisation and sustains Streptomyces hyphal growth

Abstract Bacterial chromosome topology is controlled by topoisomerases and nucleoid-associated proteins (NAPs). While topoisomerases regulate DNA supercoiling, NAPs introduce bends or coat DNA upon its binding, affecting DNA loop formation. Streptomyces, hyphal, multigenomic bacteria known for producing numerous clinically important compounds, use the highly processive topoisomerase I (TopA) to remove excessive negative DNA supercoils. Elongated vegetative Streptomyces cells contain multiple copies of their linear chromosome, which remain relaxed and relatively evenly distributed. Here, we explored how TopA cooperates with HupA, an HU homologue that is the most abundant Streptomyces NAP. We verified that HupA has an increased affinity for supercoiled DNA in vivo and in vitro. Analysis of mutant strains demonstrated that HupA elimination is detrimental under high DNA supercoiling conditions. The absence of HupA, combined with decreased TopA levels, disrupted chromosome distribution in hyphal cells, eventually inhibiting hyphal growth. We concluded that increased HupA binding to DNA under elevated chromosome supercoiling conditions is critical for the preservation of chromosome organisation.


Construction of hupA deletion strains
To construct the S. coelicolor strain lacking the hupA gene, first we constructed a E59 cosmid derivative containing hygromycin resistance cassette instead of hupA gene using primers hupA_FW and hupA_RV and PCR targeting method, which yielded E59 ΔhupA::hyg cosmid. Secondly hygromycin resistance cassette was removed by SnaBI digestion and religation to create E59 ΔhupA::scar cosmid. In this cosmid ampicillin resistance cassette in SuperCos was replaced with hygromycin resistance cassette containing oriT site, necessary for conjugation into S. coelicolor.
In order to complement hupA deletion a 769 bp long fragment containing hupA gene and its promoter sequence was amplified using hupA_FW and hupA_RV primers and then cloned into pIJ170 at KpnI site with the SLIC method yielding pIJ170 hupA plasmid. After verification with sequencing this vector was introduced into ASMK011 (ΔhupA::scar) and ASMK031 (ΔhupA::scar, TopA depletion) strains in order to obtain ASMK013 and ASMK033 strains, respectively.
HupA-FLAG construction 773 bp long fragment containing hupA gene with its promoter sequence was amplified with primers SCO_HupA_promoter_Fw and SCO-HupA-FLAG_Rv. The obtained sequence was cloned into the pGEM -SMC-FLAG plasmid at NcoI-BamHI sites using the SLIC method, replacing the SMC gene. Then fragment containing hupA-FLAG gene and promoter sequence was amplified using SLIC-hupA-FLAG_FW2 and SLIC-hupA-FLAG_RV2 primers and cloned into pIJ170 integrative plasmid at XmaJI site using the SLIC method yielding pIJ170 hupA-FLAG plasmid. This vector, after verification with sequencing, was used to transform strain ASMK011 (ΔhupA) , PS04 (TopA*) and ASMK031 (ΔhupA TopA*). Obtained colonies resistant for hygromycin were verified using Western blot for production of HupA-FLAG protein yielding the strains ASMK012, ASMK032 and ASMK034.

HupA-PAmCherry construction
First, 751 bp long fragment containing PAmCherry gene was amplified with pam_FW and pam_RV primers and cloned into pIJ170-FLAG at XhoI and XmaJI site to obtain pIJ170 PAmCherry vector. Next, a 769 bp long fragment containing hupA gene and its promoter sequence was amplified using hupA_pam_long_FW and hupA_pam_long_RV primers and then cloned into pIJ170 PAmCherry at KpnI site with the SLIC method yielding pIJ170 hupAPAmCherry plasmid. After verification with sequencing this vector was introduced into ASMK011 (ΔhupA::scar) and ASMK031 (ΔhupA::scar, TopA*) strains in order to obtain ASMK015 and ASMK035 strains respectively.
ermhupA construction First, 322 bp long fragment containing hupA gene was amplified with ermhupA_FW and ermhupA_RV primers and cloned into pIJ10257 (contains constitutive promoter erm) vector digested with XhoI using the SLIC method. After verification with sequencing pIJ10257 ermhupA vector was introduced into M145 strain yielding strain AS41.
TopAsv purification E. coli strain containing pET28topAsv was used for protein production. For protein overproduction, cells were grown to OD600 0.4 at 37°C, then isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final ∼ concentration of 0.3 mM and the culture was continued for 4 h at 37°C. The cells were collected by centrifugation, re-suspended in 50 mM NaH2PO4, pH 8.1, 300 mM NaCl with 20 mM imidazole and sonicated. Fast protein liquid chromatography (FPLC) system with HisTrap HP columns (GE Healthcare) was used to purify recombinant proteins from cell lysate, followed by desalting using Zeba Spin Desalting Column (Thermo Scientific) equilibrated with 50 mM NaH2PO4, pH 8.1, 300 mM NaCl, 10% glycerol buffer. Protein samples were stored in -80°C. B. TopA relaxation assay followed by incubation with HupA. Two hundred nanograms of supercoiled plasmid pUC19 was initially incubated with TopA (30-120 nM) for 15 minutes, followed by the addition of HupA (0-8 μM) and incubation for 15 min at 20°C. Topoisomers were resolved without deproteinization by agarose gel electrophoresis.
The positions of the supercoiled and relaxed topoisomers are indicated.  Strains were cultured in YEME/TSB medium for 24h. Each sample was performed in triplicate. Statistical analysis was performed using ANOVA with Tukey post-hoc test, statistical significance is given against the wild type strain.

Fig S4
Comparison of AT% percent calculated for ChIP-seq HupA binding sites identified by edgeR (left) and MACS3 (right) and the same number of random S. coelicolor sequences of similar length, p-values calculated by two-sided Wilcoxon test is shown on the plot.  A. Snapshots from the time-lapse analysis of the ParB-EGFP (green) and DnaN-mCherry complexes (red) in vegetative hyphae of the control strain (AK101) and TopA-depleted strain (TopA*, AS11). The fluorescence images are next to the DIC images (grey). Scale bar 1 μm.
C. Average distance between the ParB complexes after duplication over time in the control strain (AK101, black) and TopA-depleted strain (TopA*, AS11, red). Lines show the linear model with 95% confidence intervals.
D. Percentage of hyphae in which duplicated ParB complexes could be detected at the indicated time after replisome appearance in the control strain (AK101, black) and TopA-depleted strain (TopA*, AS11, red). Error bars show 95% confidence intervals.

Fig. S8
A. Snapshots from the time-lapse analysis of the DnaN-EGFP (green) in germinating spores of the control strain (J3337) and TopA-depleted strain (TopA*, AS07). The fluorescence images are overlayed with the DIC images (grey). Scale bar 1 μm. B. Number of DnaN-EGFP complexes divided by hyphae length over time for the control strain (J3337, black, 30 hyphae) and TopA-depleted strain (TopA*, AS07, red, 30 hyphae). Shown curve was fitted using loess algorithm.